Substrate-Specific Coupling of O2Activation to Hydroxylations of Aromatic Compounds by Rieske Non-heme Iron Dioxygenases

Abstract

Rieske dioxygenases catalyze the initial steps in the hydroxylation of aromatic compounds and are critical for the metabolism of xenobiotic substances. Because substrates do not bind to the mononuclear non-heme FeIIcenter, elementary steps leading to O2activation and substrate hydroxylation are difficult to delineate, thus making it challenging to rationalize divergent observations on enzyme mechanisms, reactivity, and substrate specificity. Here, we show for nitrobenzene dioxygenase, a Rieske dioxygenase capable of transforming nitroarenes to nitrite and substituted catechols, that unproductive O2activation with the release of the unreacted substrate and reactive oxygen species represents an important path in the catalytic cycle. Through correlation of O2uncoupling for a series of substituted nitroaromatic compounds with 18O and 13C kinetic isotope effects of dissolved O2and aromatic substrates, respectively, we show that O2uncoupling occurs after the rate-limiting formation of FeIII-(hydro)peroxo species from which substrates are hydroxylated. Substituent effects on the extent of O2uncoupling suggest that the positioning of the substrate in the active site rather than the susceptibility of the substrate for attack by electrophilic oxygen species is responsible for unproductive O2uncoupling. The proposed catalytic cycle provides a mechanistic basis for assessing the very different efficiencies of substrate hydroxylation vs unproductive O2activation and generation of reactive oxygen species in reactions catalyzed by Rieske dioxygenases.